Method for preparing vanillin and composition therefor

ABSTRACT

A new radioenzymatic assay for catecholamines utilizing the catechol-0-methyl transferase transfer of a methyl group from a labeled methyl donor to the catecholamines followed by isolation of the 0-methylated( 3  H)catecholamine and the subsequent measurement of radioactivity.

This is a division of co-pending Ser. No. 950,863 filed Oct. 12, 1978and now U.S. Pat. No. 4,242,222 which is a division of Ser. No. 681,999,filed Apr. 30, 1976, now abandoned.

BACKGROUND OF THE INVENTION

Diagnostic tests have become an indispensible tool of a physician'sarmamentarium. Accompanying many diseases and conditions are subtlechanges in the body's physiology, particularly in concentrations ofcompounds associated with the condition. The catecholamines are a groupof these compounds. They are identified by the catechol nucleus ##STR1##wherein R is the amine function of the particular compound. Thesignificant catecholamines found naturally in the body are epinephrine,R is ##STR2## hereafter referred to as E; norepinephrine, ##STR3##hereafter referred to as NE: and dopamine, R is --CH₂ CH₂ MH₂, hereafterreferred to as DA. These catecholamines are found in various tissues ofthe body, including, inter alia, blood, urine, cerebrospinal fluid, andbrain tissue. The quantities of these compounds are extremely small. Forexample, in blood serum E and NE are present in picogram (10⁻¹²)quantities.

In recent years there has been substantial interest in measuringcatecholamine levels in mammalian systems. Catecholamine levels aresignificantly increased when pheochromocytomas are present. Thecatecholamine levels are influenced by the presence of other tumors,such as neuroblastoma, which affect the central nervous system.Knowledge of catecholamine levels is of significance in the diagnosisand management of hypertension, coronary disease, angina pectoris, acutemyocardial infarction, and diabetes mellitus.

An early method for measuring catecholamine levels in mammalian systemswhich was sufficiently sensitive to reach the submicrogram range was thespectrofluorimetric assay of Von Euler and Floding (Acta Physiol. Scan.33: Suppl. 118, 45, 1955). This assay was based on the oxidation of NEand/or E with ferricyanide or iodine to the correspondingtrihydroxyindole which in alkaline solution yields a highly fluorescentnoradrenolutine or adrenolutine. NE or E must be first isolated from themammalian system, for example, blood serum or plasma, the supernatant ofdeproteinized tissue homogenate or cerebrospinal fluid, before the assaycan be done. DA is oxidized by iodine to form a dihydroxyindole. Thefluorophore produced by this derivative is different from that of NE orE and is the basis for the fluorimetric assay (Carlson and Waldeck, ActaPhysiol. Scand. 44: 293, 1958). As with NE and E, DA must also beisolated from the mammalian system prior to the assay.

Isolation of the cotecholamines from the superratant of deproteinizedtissue homogenate, the blood plasma or the biological fluid is generallydone by one of two techniques. The first and most widely used isolationtechnique is the adsorption of catecholamines onto neutral alumina at pH8.4-8.6. Catechol containing compounds are adsorbed by alumina andcatechol compounds from the mixture are discarded. Catecholes areremoved from the alumina by acid elution.

The second isolation technique employs cation exchange chromatography.Due to the ionization of these amines at acidic pH, ME, E and DA can bebound to the exchange resin and then eluted preferentially by increasingacid strength.

Both of these isolation processes are quite lengthy and generally do notyield fractions which are specific for a particular catecholamine.Moreover, the spectrofluorimetric assay systems are relativelyinsensitive for the needs of the investigator. For NE and E asensitivity of approximately 50-500 nanograms is obtained. Althoughrefinement of this method has led to techniques that are sensitive toquantities as small as 2 to 3 nanograms, the accuracy of assays at thislevel of sensitivity are poor, see Engelman et al. Am. J. Med. Sci.,255, 259 (1962). The sensitivity of DA is approximtely 500 nanograms.Even though this assay is relatively insensitive, all the commerciallaboratories but one use the spectrofluorimetric assay or someadaptation of it for measuring catecholamines.

Another method of commercially measuring catecholamine levels ispotentially available. The enzyme catechol-O-methyl transferase,initially reported and characterized by Axelrod and Tomchick, J. Biol.Chem., 233, 702, 1958, and hereafter referred to as COMT, is an enzymewhich transfers a methyl group from a donor molecule to a catecholnucleus thereby forming a 3-methoxy moiety depicted below in FIG. 2wherein R is the amine functionality. ##STR4##

Later investigators using radiotracers and other techniques includingchromatography have related the radiolabeled quantities of metanephrine,hereafter referred to as MM, normetanephrine, hereafter referred to asMMM and methoxytyramine, hereafter referred to as MEOT to the initialquantities of E, NE and DA in the mammalian system. The chemicalstructures of MM, MMM, and MEOT are shown in FIG. 3. ##STR5##

In 1968 Engelman, et al., Am. J. Med. Sci. 255, 259 (1968) utilized thedouble isotope dilution derivative technique with COMT to assay forcatecholamines, i.e., epinephrine and norepinephrine, in biologicalspecimens. An internal tracer, 7-H³ -norepinephrine was employed withthe methyl donor S-adenosyl-L-methioninemethyl-¹⁴ C. Prior to assay theurine and blood samples were pretreated to separate the catecholamines.The urine was chromatographed over alumina and the blood samplechromatographed over a cation-exchange resin. After incubation of thechromatographed samples in the enzymatic system, the incubation wasstopped and the resulting solution treated and chromatographed over acation exchange resin. The methanephrines were eluted and oxidized tovanillin with periodate. The vanillin was extracted, purified andcounted in a liquid scintillation medium. The typical complex doubleisotope measurement and calculation was required to find the quantity ofcatecholamine present.

Engelman followed up his earlier work with another publication, Engelmanet al., Circulation Research, 26, 53 (1970). Modification of his earlierwork now allowed the differential measurement of E and NE in the samesample of 5 to 10 ml. human blood plasma. The same double isotopetechnique was employed as in the previous publication except that the MMand MMM are separated with thin layer chromatography. Tritium labeledtracer quantities of both E and NE were added along with 100 μg. of bothMM and MMM. Prior to assaying, both plasma and urine samples werechromatographed over cation exchange resins. Ethylenediaminetetraaceticacid was added to the plasma sample prior to the chromatographic step.The sensitivity of the assay, as measured by the value which is twicethat of the blank, was 250 picograms. Plasma E values cannot be measuredvery accurately at the lower end of the normal range.

In Passon and Peuler, Analytical Biochemistry, 51, 618, (1973) E and NEwere assayed using COMT, followed by separation of MM and MMM bychromatography, and thereafter oxidation to vanillin. The blood serum orplasma was used directly without any prior chromatography. Only a singleisotope, S-adenosyl-methionine(³ H)methyl was used. The tritiated methyldonor was diluted with cold methyl donor. Tracer labeled ¹⁴ C compoundswere tried but were not helpful in reducing problems such as an accurateestimate of recovery of the catecholamine substrate. At concentrationsbelow 10 μM of cold S-adenosylmethionine, the blank increasedsubstantially. Assay techniques were otherwise similar to those used byEngelman, et al. and others. The sensitivity of the assay was 170 pg.for NE and E, thereby allegedly allowing the measurement of NE and E inless than 1 ml. of human serum.

Coyle and Henry, J. of Neurochemistry, 21, 61 (1973)separatednorepinephrine from dopamine in deproteinized brain tissuehomogenates by using COMT and S-adenosyl-methionine(³ H)methyl. Afterthe addition of non-radioactive carrier quantities of MMM and MEOT, theincubate was extracted by an organic solution and the extractrepartitioned into aqueous hydrochloric acid. The aqueous phase waswashed with the organic solvent. Vanillin-(³ H) was produced byperiodate cleavage ³ H MMM. The vanillin-(³ H) was extracted into anorganic solvent and eventually counted in a liquid scintillation medium.The MEOT was extracted from the aqueous periodate phase with a boratebuffer and a 3:2 (v/v) toluene-isoamyl alcohol solvent system andcounted with a liquid scintillator.

Christensen, Scand. J. Clin. Lab. Invest. 31, 343 (1973) determined thelevel of DA in plasma by a double isotope technique utilizing the COMTprocedure. Prior to the enzymatic methyl transfer, the catecholamineswere isolated from plasma utilizing aluminum. The COMT and the samplewere incubated with S-adenosylmethioninemethyl (¹⁴ C) and tracerquantities of tritium labeled DA. After oxidation of MM and MMM withperiodate to vanillin, the MEOT is separated and analyzed in a liquidscintillator. The DA concentration was calculated on a computer from thecounts of ¹⁴ C and ³ H.

Cuello et al. in 1973 analyzed deproteinized brain tissue extracts forDA in the presence of NE. COMT and tritium labeled S-adenosylmethioninewere mixed with cold S-adenosylmethionine or ¹⁴ C S-adenosylmethionine.After extracting the incubate with an organic solvent, the organicsolvent is back extracted into hydrochloric acid and thenchromatographed.

In 1975, Yamaguchi et al., Circulation Research, 36, 662 (1975) assayedfor endogenous catecholamines in blood with ethylene glycolbis-(aminoethyl ether)-N,N'-tetraacetic acid (hereafter referred to asEGTA) present in the incubation mixture.

Champlain et al., Circulation Research, 38, 109 (1976) disclosed thatcalcium ion was known to inhibit the reaction of COMT from work done byAxelrod and Tomchik. EGTA is a selective chelator of calcium as opposedto magnesium and restores the activity of the enzyme. However, a laterpublication, Weinshilbaum, et al., Biochemical Pharmacology, 25, 573(1976) states that the possibility that other cations might inhibit theCOMT reaction in the presence of optimal concentration of magnesium hadnot been studied to date.

A new system for assaying catecholamines has now been developed,utilizing the basic COMT system of the past. Catecholamines content ofmammalian systems can now be assayed to a sensitivity of 5 picograms forNE and E and 12 picograms for DA. The assay is precise and unusuallyselective. Furthermore, the assay can be performed rapidly by laboratorypersonnel having minimal advanced technical training. The assay ispresently being used on a successful commercial basis by The LaboratoryProcedures Division of The Upjohn Company.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with this invention there is a method for analyzingundifferentiated endogenous epinephrine and norepinephrine levels in amammalian system selected from the group consisting of the supernatantof the deproteinized tissue homogenate, blood plasma and serum, andbiological fluid which comprises

a. bringing together an aliquot of the supernatant of the deproteinizedtissue homogenate, blood serum or plasma, or biological fluid, with theenzyme catechol-O-methyl transferase, the tritium labeled methyl donorS-adenosyl-L-methionine(³ H)methyl substantially free of unlabeledmethyl donor, a cation of oxidation number +2 which allows the methyltransfer to proceed, a compound which stabilizes the enzyme-substratesystem, and an agent which preferentially removes calcium ions frominterference with the enzymatic reaction,

b. incubating together the components of "a" for a time, temperature andpH sufficient to O-methylate (³ H) substantially all of the epinephrineand norepinephrine;

c. extracting the O-methylated (³ H) epinephrine and norepinephrine withan organic solvent in which the O-methylated (³ H) epinephrine andnorepinephrine are preferentially soluble;

d. repartitioning the O-methylated (³ H) epinephrine and norepinephrineinto an aqueous acid of sufficient strength to protonate the amine;

e. oxidizing O-methylated (³ H) epinephrine and norepinephrine tovanillin-³ H.

f. extracting vanillin-³ H from the aqueous solution with an organicsolvent in which the vanillin-³ H is preferentially soluble, and

g. counting the radiation emitted from the vanillin-³ H.

A further aspect of the invention is the method for differentiallyanalyzing the levels of epinephrine and/or norepinephrine obtained froma mammalian system selected from the group consisting of the supernatantof deproteinized tissue homogenate, blood serum or plasma, andbiological fluid which comprises performing the same assay steps asabove with the additional step of separating the O-methylatedepinephrine and O-methylated norepinephrine by chromatographic means.This separation step is performed after the repartitioning and beforethe oxidation of O-methylated (³ H) epinephrine and/or norepinephrine tovanillin-³ H.

Another aspect of the invention is the method for assaying the levels ofdopamine present in the sample. In the undifferentiated epinephrine andnorepinephrine assay, the aqueous oxidation phase remaining after thevanillin extraction is extracted at a basic pH with an organic solventand the MEOT (³ H) counted. In the differential assay for epinephrineand norepinephrine, the dopamine is analyzed in the same manner as inthe undifferential assay when the solvent system used to separate theO-methylated (³ H) epinephrine and O-methylated (³ H) norepinephrine isacidic. If the solvent system for separating the O-methylated (³ H)epinephrine from the O-methylated (³ H) norepinephrine is basic, the ³ HMEOT is also separated in the chromatographic system. The ³ H MEOT iseluted and counted.

Another aspect of the invention is the use of an agent to selectivelyremove calcium ions from interference with the COMT enzymatic reaction.

A further aspect of the invention is a method for increasing theintrinsic activity of COMT by using EGTA in combination with COMT.

Another aspect of the invention is a method for purifying rat liver COMTutilizing a further ammonium sulfate fractionation. A new COMTcomposition resulting from this purification is a further aspect of theinvention.

A still further aspect of the invention is the purification ofvanillin-³ H in a scintillation vial employing an acid wash and a methodfor assaying the radioactivity in such a vial.

Another aspect of the invention is the oxidation of metanephrine-³ H ornormetanephrine-³ H to vanillin-³ H with periodate in the presence ofsilica gel and the composition comprising those components.

A further aspect of the invention is a new basic solvent system used forthin layer chromatography.

Another aspect of the invention is the presence of an aromatic L-aminoacid decarboxylase, particularly DOPA decarboxylase, inhibitor in theincubate. A compound of this type inhibits the production of dopamineduring this incubation period.

A further broad aspect of the invention is the assemblage of theessential components of the assay into a kit which may be used bylaboratories.

Other aspects of the invention will become clear as the description ofthe invention continues.

DETAILED DESCRIPTION OF THE INVENTION

The catecholamines are found throughout various mammalian systems. Asstated previously, these compounds have in common the catechol nucleusand an amine function. Examples of various mammals in whichcatecholamines can be assayed include human, horse, cattle, dog, cat,rat, mouse, rabbit, and other animals generally used in scientificlaboratories for the study of circulatory and myocardial problems.Catecholamines are found in various systems throughout the mammal.Circulating levels of catecholamines are found in the blood. Otherbiological fluids in the body, for example, urine, cerebrospinal fluidand lymphatic fluids also have significant levels of catecholamine.Tissue samples which can be employed in the assay system is any tissuewith sympathetic innervation, such as vascular tissue, liver, adrenaland kidney; brain tissue; tumor containing tissue and the like. Whentissue is being assayed, the sample is broken down by any commontechnique such as homogenizing, tissue press under pressure, andsonication. It is the supernate of this homogenate which is thenemployed in the assay. In order to maintain the integrity of the system,an acidic pH should be employed. When liquid systems such as bloodfractions or other biological fluids are being employed, no preliminaryisolation such as alumina or cation exchange chromatography need beemployed. Rather the fluid sample per se can be employed.

The size of the sample to be assayed varies substantially depending uponthe quantities of catecholamines present. Because of the sensitivity ofthe assay, blood serum or plasma samples as low as 10 μl. or even lessmay be readily assayed. Urine samples generally have to be diluted sincethe quantities of catecholamines present in urine are significantlyhigher than in blood samples. It should also be noted that tissuesamples containing phenochromocytoma will have significantly highercatecholamine levels. The upper level of the volume of the mammaliansystem being assayed can go as high as one milliliter, if desired. Thevolume levels required are generally so minute that small laboratoryanimals can be readily assayed without significant problems. Thequantity of catecholamine present in each sample aliquot can vary fromabout 20 to about 10,000 picograms of E or NE for the undifferentiatedassay and from about 3 to about 5,000 for the differential assay for E,NE, and DA. Depending upon the picogram concentration, a general samplealiquot of the system to be assayed is from about 10 to about 50 μl.

As noted previously, COMT is an enzyme which has a general activity forthe O-methylation of a catechol nucleus. Although the assay methodologydisclosed in this invention is quite specific for endogenouscatecholamines, it does not differentiate catechol nuclei from othersources. For example, many drugs administered to mammals, specificallyhumans, have catechol nuclei or are precursors of metabolites havingcatechol nuclei. Illustrative examples of such drugs and/or metabolitesare α-methyl norepinephrine, α-methyldopamine, isoproterenol,α-methyldopa, and other like compounds. If a mammal has received suchdrug treatment, the results of this assay utilizing COMT would be undulyhigh. Consequently, mammals receiving such compounds are inappropriatesubjects for this assay.

The enzyme COMT is found in a wide variety of mammals. Examples of suchmammals include rat, cow, pig, mouse, guinea pig, cat, rabbit and man.The enzyme is widely dispersed throughout the systems, for examples,liver, kidney, spleen, small intestine, brain the like. The preferredspecies is the rat. The preferred organ from which the enzyme isisolated is the liver. The methods of isolation are known throughout theart. The usual steps are followed in isolating the enzyme, with theexception that a further ammonium sulfate fractionation is employed.This step, not heretofore practiced in the art, involves resuspendingthe 30-50 or 55% ammonium sulfate precipitate in a 55% saturatedammonium sulfate solution, separating the phases and proceeding with theusual isolation steps. The addition of this extra fractionation stepbrings about a substantial reduction in the blank value.

S-adenosyl-L-methionine-(³ H) methyl is the labeled methyl donoremployed in the enzymatic O-methylation. This tritium labeled methyldonor can be made by art known radiochemical methods and is commerciallyavailable from New England Nuclear Corp. and Amersham-Searle. Thetritium label is at the methyl position. It has now been found that thismethyl donor should be present in the incubation mixture at aconcentration ≧about 4 μM for maximal activity and maintenance of thecatecholamines as the rate determining concentration in theO-methylation. In order to produce the excellent sensitivity of thisassay, the labeled methyl donor is essentially free of unlabeled, coldmethyl donor S-adenosyl-L-methionines. The addition of unlabeled coldmethyl donor makes the assay less sensitive to the catecholamines. If aless sensitive assay is desired, this may be achieved by addingunlabeled, cold S-adenosyl-L-methionine to the assay incubate. Thetritium labeled S-adenosyl-L-methionine-(³ H) methyl should be preparedwith maximum radioactivity for maximum sensitivity of the assay.Radioactivity greater than or equal to 8 Ci/mmol can be employed.Preferred radioactivity is from about 10 to about 15 Ci/mmol.

In order for the enzymatic system to be active, at least one of a numberof cations of oxidation number +2 must be present in the incubate.Cations of oxidation number +2 which can be used are magnesium, cobalt,and manganese. Magnesium is preferred. The metal can be present as asalt, for example, magnesium chloride. A concentration of cation of fromabout 10 to about 100 mM, preferably about 25 to about 35 mM, is presentin the incubation mixture.

Also present in the incubate is a compound which stabilizes theenzyme-substrate system. This compound maintains the integrity of thesystem by preventing oxidation of the catecholamines and assists in thecontinued activity of the enzyme during the incubation. Compounds whichcan be effectively employed to stabilize the enzyme-substrate system areglutathione, dithiothreitol, ascorbic acid, sodium metabisulfite,mercaptoethanol, cysteine, and the like. Glutathione is preferred.Concentration of the stabilizer in the incubate is from about 1 to about10 mM, preferably about 2 to about 4 mM. When ascorbic acid is employed,care must be taken to avoid an excess since an inhibition of enzymaticactivity can occur.

A further component of the incubate is an agent which preferentiallyremoves calcium ions from interference with the enzymatic conversion.Calcium ions in the presence of activator ions such as magnesium, cobaltor manganese inhibit the enzymatic O-methylation. Consequently, it isextremely advantageous to have present in the incubate an agent whichselectively removes calcium ions instead of activator ions fromparticipation in the enzymatic conversion. Agents which accomplish thisfunction are illustratively EGTA, Chelax®, a resinous substanceobtainable from Biorad Laboratories, sodium oxalate, dyes which chelatecalcium ion selectively, for example, Murexide®, and other like agents.EGTA is preferred. Since the agents may differ in their selectivity forcalcium ion with respect to the activator ion, extra activator ion maybe needed and should be added if necessary. These agents are present insufficient quantity so as to allow the enzymatic conversion to proceedas rapidly and as efficiently as possible. For example, concentrationsof from about 5 to about 25 mM of EGTA in the incubation mixture can beused.

It has also been found that EGTA brings about an increase in theintrinsic activity of COMT. When added to the blank of the incubate,that is, all components of the incubate but the mammalian system, EGTAbrings about an increase in COMT enzymatic activity as measured by theamount of O-methyl(³ H)catecholamine formed.

All the quantities of the incubate are present in quantities which allowthe catecholamine concentration to be the rate limiting factor. Theenzyme, methyl donor, cation, stabilizing compound and agent whichselectively removes calcium ions are present in quantities in excess ofthose quantities necessary to bring about the timely conversion of thecatecholamines to O-methylated(³ H)catecholamines. All, or essentiallyall, of the catecholamines present in the mammalian system to be assayedare converted during the incubation period.

The enzymatic incubation is carried out in standard laboratory equipmentfor a time and at a pH and temperature which allows the enzymaticconversion to go to completion. The pH of the incubation should bemaintained between about 7 and about 10. The preferred pH range is fromabout 8 to about 9. Buffer solutions which are suitable for theincubation are tris, phosphate, or glycine/NaOH. The temperature of theincubation can be from about 25° C. to about 45° C., preferably fromabout 35° C. to about 40° C. The incubation is allowed to proceed for aperiod of from about fifteen minutes to about two hours, preferably fromabout forty to about sixty minutes.

The incubation is stopped by any of a variety of standard means. Forexample, the temperature can be lowered or elevated or the pH can beraised or lowered. It is preferred to stop the incubation by a loweringof the temperature and the introduction of a higher pH accompanied by amagnesium, cobalt, or manganese binding agent. If desired for economy,time factors, or personal preference, the incubation need not be stoppedbefore the O-methylated(³ H)catecholamines are selectively removed fromthe incubate.

The stopping solution can also contain carrier quantities of MM, MMM andMEOT. Since there is such a small quantity of catecholamines present inthe sample, the carrier compounds are advantageously added to facilitatethe recognition and partitioning of the sample catecholamines throughoutthe assay. The carrier compounds are generally present as theirhydrochloride acid addition salts since this form of the compound isthought to be more stable. In the succeeding assay step, the free baseis formed. The carrier compounds are preferably added in the stoppingsolution prior to the organic solvent extraction of the incubate,although they can be added to the incubate initially, depending upon theeuilibrium in the COMT transfer. If the incubate is not stopped, thecarrier quantities of MM, MMM and MEOT should still be added to theincubate prior to the preferential organic solvent. A concentration ofcarrier of from about 2 to about 4 mM can be employed.

The O-methylated(³ H)catecholamines are now selectively removed from theincubate by solvent extraction. An organic solvent which extracts theMM, MMM and MEOT from the incubate is employed. It is, of course,preferred to have an organic solvent in which the O-methylatedcatecholamines are preferentially soluble, that is, leaving as much ofthe incubate components, side reaction products, and other extraneousmatter in the aqueous incubate. Any organic solvent which issubstantially immiscible with water can be used. Illustrative examplesof organic solvents which can be employed are the higher alcohols suchas butanol, isoamyl alcohol, hexanol, esters such as ethyl acetate,ethers such as diethyl ether, tetrahydrofuran, dioxane, aromatics suchas benzene, toluene, and the xylenes. Preferred organic solvents areisoamyl alcohol, butanol, toluene, and the xylenes. More preferred arecertain mixtures of organic solvents, specifically a 35-75% volume tovolume, mixture of toluene:isoamyl alcohol, preferably 50-65%toluene:isoamyl alcohol.

The organic extract of O-methylated(³ H)catecholamines is nowrepartitioned into an aqueous acid of sufficient strength to protonatethe amine function of the catecholamines. Examples of such acids areformic, acetic, propionic, hydrochloric, sulfuric, phosphoric and thelike. When performing the assay for the analysis of total,undifferentiated E and NE, any of these acids can be used. However, whenthe differential assay for E and NE is performed, it is preferable touse an acid which has a relatively low boiling point and which leavesessentially no residue upon evaporation, for example, formic, acetic andpropionic acid, preferably acetic acid. The differential assay requiresa chromatographic separation step of MMM from MM. The presence of theacid addition salt of MM or MMM interferes with the separation of MMfrom MMM and each catecholamine from other constituents of the sample.This repartition can be repeated several times for an increasedpartitioning effect.

We have found that when the aqueous phase is denser than the organicphase, separation of the two phases is facilitated by freezing theaqueous phase. Freezing shortens the assay time and allows for acleaner, more precise separation of the organic phase from the aqueousphase.

If the total undifferentiated values for E and NE are desired, thefollowing separation step of MM from MMM is not done. Rather, theaqueous acid extract is preferentially dried down, taken up in basicsolution and the MM and MMM oxidized to vanillin.

When the differential assay for E and NE is performed, MM is separatedfrom MMM at this point. The usual means of separating the O-methylatedcatecholamines is by chromatography, for example, paper and thin layer,preferably thin layer chromatography. The aqueous acid extractcontaining the O-methylated catecholamines is preferably taken todryness and then solubilized in an organic solvent such as 95% ethanol.The ethanolic solution is applied to a chromatographic plate coveredwith a thin layer of adsorbent, for example, siliceous materials. Thethickness of the layer of adsorbent is that generally used in the art,for example, 50 to 750μ, preferably 200-300μ. Silica gel is preferred.The solvent system used for the separation can be any of the systemsknown to separate MM from MMM. However, it is preferred to use withsilica gel a solvent system of composition 60 isopropyl alcohol, 20n-butanol, 19 water, and 1 formic acid, as volume to volume percentages.When using this solvent system, MM has an R_(f) of about 0.59 and MMM anR_(f) of about 0.7. The MEOT does not separate from the MMM. Theseparation of MM from MMM occurs rapidly and distinctly. Another solventsystem when used in conjunction with silica gel brings about theseparation of MEOT from MMM. The basic solvent system of 6 partst-amylalcohol:2 parts benzene:3 parts methylamine, volume to volume,brings about a separation on silica gel wherein MEOT is furthest fromthe origin, MM is closer to the origin, and NMN is closest to theorigin. This latter solvent system allows for the ready analysis of DAvia the radioemission of the separated MEOT.

The basic solvent system employed in the above thin layerchromatographic step is novel. The essential components of the systemare alcohol to methylamine in a ratio of about 1:1 to about 5:1 volumeto volume alcohol to methylamine. The methylamine is 40% in water. Thealcohol is a higher alcohol such as t-amyl alcohol, i-amylalcohol,n-butanol and the like. A non-polar, lipophilic organic compound mayalso be present in the solvent system. The presence of such a solventincreases the velocity of migration and spot resolution. It may bepresent in as high a quantity as is consonant with a one-phase solventsystem. Illustrative examples of non-polar lipophilic organic solventswhich can be employed are benzene, toluene, m-xylene and p-xylene.

At this point in the assay, the spot containing MN is eluted from thesilica gel with a solvent such as ammonium hydroxide, and then extractedwith an organic solvent or organic solvent system as previouslydisclosed with respect to extracting the incubate.

The radiolabeled compounds are then counted. When using the basicsolvent system in the chromatographic separation, the MEOT and NMN canbe analyzed in the same manner. However, it is preferred to oxidize theMN and NMN obtained from either the acidic or basic solvent separationsystem to vanillin. This oxidation gives greater specificity to theassay since only the β-hydroxy catecholamines are subject to thisoxidation. This step separates other labeled catechol nuclei not bearinga β-hydroxy group. Also, since in the acidic solvent system the NMN andMEOT are not resolved, the oxidation of NMN to vanillin is necessary.When only assaying for total or undifferentiated E and NE, the oxidationis also necessary to separate the MEOT from the MN and NMN.

The oxidation of the β-hydroxy O-methylated catecholamine isaccomplished with standard reagents and conditions. After elution of theMN or NMN from the adsorbent, the MN and NMN is contacted with anoxidizing agent such as sodium metaperiodate at a pH of from about 7 toabout 12 at a temperature of from about 0° to about 50° C. for a periodof from about two to about thirty minutes. The MN and NMN should bequantitatively oxidized to vanillin. The temperature of the reaction ispreferably from about 0° to about 40° C., and the time of the reactionis preferably two to ten minutes.

It now has been found that the periodate oxidation can be carried out inthe contact presence of silica gel. This increases the amount of assaysensitivity, accuracy and precision since the oxidation step may now becarried out in a single container. The silica gel band containing MN orNMN is scraped off the plate and placed into a tube, the MN or NMNeluted with a basic solvent such as ammonium hydroxide and sodiummetaperiodate added. The oxidation to vanillin occurs smoothly and withno difficulty in the contact presence of silica gel.

The oxidation is completed or stopped. The stopping substituent isgenerally a glycerol solution. If the pH of the system is not already ator below 7, the pH of the system is reduced to <7 with acid. The natureof the acid employed is not significant. Hydrochloric, sulfuric,phosphoric, formic, acetic or propionic are illustrative of the acidswhich can be employed.

The vanillin is now extracted into an organic solvent which issubstantially immiscible with the aqueous fraction. This organic solventis preferably relatively non-polar so as to effect a good separationbetween vanillin and relatively polar side products. Examples of suchsolvents are illustratively benzene, toluene, m-xylene, p-xylene,ethylbenzene, isopropylbenzene, esters such as ethyl acetate, andisopropylacetate, and ethers such as diethyl ether and 1,4-dioxane. Whenthe aqueous phase is denser than the organic phase, separation of thetwo phases is facilitated by freezing the aqueous phase and removing theorganic phase.

The vanillin-³ H is now counted. Although various radioemission countingtechniques can be employed, such as planchet counting, a liquidscintillation counting technique is preferred. The particular liquidscintillator employed is not significant as long as it will emit aquantum of light when exposed to a β-ray emitted from tritium. Examplesof suitable scintillators are p-terphenyl; 2,5-diphenyloxazole;2-phenyl-5-(4-biphenylyl)-1,3,4-oxadiazole,2,5-bis-2-(5-t-butylbenzoxazolyl)thiophene;2,5-diphenyl-1,3,4-oxadiazole; 2-(4'-biphenyl)-6-phenyl-benzoxazole;1,4-bis-2-(5-phenyloxazolyl)benzene. The scintillator is solubilizedwithin an organic solvent, the combination of scintillator and solventhereafter referred to as the liquid scintillation medium. The organicsolvent containing vanillin is added to the liquid scintillation mediumand counted. A preferable method of extracting the vanillin-³ H is touse the iquid scintillation medium as the extractant. The specificity ofthe assay can be further improved by contacting the vanillin containingorganic solvent with an aqueous acid, thereby removing additional watersoluble impurities. Although this extraction can be done prior to theaddition of the vanillin containing organic solvent to the liquidscintillation medium, it is preferred to have the aqueous acid phase incontact with the liquid scintillation medium. In this manner, the numberof separate transfers and phase separations is minimized. When thevanillin is added to the liquid scintillation medium, the vanillinremains in the organic layer and is counted by the scintillator.However, any tritium labeled contaminant extracted into the aqueous acidphase is not counted since the scintillator is in the organic phase andthe tritium emission, β-ray, is stopped by the aqueous phase andinterface. The liquid scintillation medium should be selected so thatthere is little or no scintillator soluble within the aqueous phase.

If DA is desired to be assayed as well, the pH of the aqueous periodatephase is raised to above 7, preferably 9.8 to 10.1, and MEOT extractedwith an organic solvent as used in the vanillin extraction. Counting ofthe radioactive emissions from MEOT-³ H is achieved in a similar manneras to vanillin-³ H.

Concurrently with the mammalian system having the unknown catecholaminelevel, a blank sample having all the incubate components except themammalian system is assayed in the same manner. The sensitivity of theassay is determined as the level of catecholamine which has double thecounts of the blank. The standard curve for the assay is obtained byassaying known quantities of E, NE or DA alongside the unknown andblank. The counts obtained with the known concentration of E, NE, or DAis plotted against the concentration. The unknown concentration ofcatecholamine is then determined by the number of counts emitted withreference to the standard curve.

In this manner, we have been able to attain sensitivities of 3 pg. forE, 5 pg. for NE, 12 pg, for DA, and 5 pg, for the total undifferentiatedE and NE. An accuracy of 5-12% coefficient of variation is obtained frommammalian systems of extremely small sample size. The standard showsfirst order kinetics for a range of from about 3 to at least 3000 pg.The assay system is versatile, rapid and commercially useful.

Following are specific examples of the assay. The examples are intendedto be only illustrative and not limiting of the invention conceptsembodied in this patent application.

EXAMPLE 1

A. Preparation of Catechol-O-methyl Transferase

The enzyme preparation generally follows the method of Axelrod andTomchick, J. Biol. Chem. 233, 702, 1958.

Rats are sacrificed. The livers are removed, rinsed in cold isotonic KCl(1.15%) blotted dry, weighed and homogenized in four volumes of isotonicKCl. The homogenate is centrifuged for thirty minutes at 78,000 g. Thesupernatant is decanted and the pH of this solution is adjusted withstirring to pH 5 with the dropwise addition of 1 N acetic acid. Afterstanding in the cold, this solution is centrifuged to remove thesuspension which forms.

The resulting supernatant is decanted and ammonium sulfate (AmSO₄) isadded slowly to bring the AmSO₄ concentration to 30% saturation. Aftercentrifugation the supernatant is decanted and AmSO₄ is added to raisethe concentration to 55% saturation. The solution is centrifuged toproduce the 30-55% AmSO₄ fraction.

The 30-55% AmSO₄ residue is now dissolved in 1 mM phosphate (PO₄)buffer, pH 7.4 containing 0.1 mM dithiothreitol (or 0.2 mM glutathione)and AmSO₄ is again added slowly to bring the AmSO₄ concentration to 55%of saturation. This additional AmSO₄ fractionation provides an enzymepreparation which provides a lower blank. After centrifugation theresidue is again dissolved in the above PO₄ buffer and dialyzed against4 changes of the same buffer. This dialysis usually takes 14-18 hours toperform.

The activity of the enzyme preparation is determined and the volume isadjusted for optional use in the catecholamine assay. Proteinconcentration of the enzyme preparation is also determined.

Due to the presence of aromatic-L-amino acid decarboxylase activity inthis preparation, a decarboxylase inhibitor, such as benzyloxyaminehydrochloride or 3-hydroxy-4-bromobenzyloxyamine phosphate, is addedinto the enzyme preparation at a concentration of 10⁻³ M. Incubateinhibitor concentration is 10⁻³ to 10⁻⁵ M.

B. Solutions

The following solutions are prepared:

1. Stabilizing Solution--0.5 ml. total volume

10 mM reduced glutathione in 0.01 N HCl

2. Standards Solution--0.5 ml. total volume

Norepinephrine, epinephrine and dopamine dissolved in stabilizingsolution to final concentration of 100 μg. of each base/ml.

3. Buffer Solution--2 ml. total volume

A solution containing 1000 mM Tris(hydroxymethyl)aminomethane, 100 mMEGTA and 300 mM magnesium chloride.

4. S-Adenosyl-L-methionine-³ H methyl--0.4 ml. total volume

Specific activity >8 Ci/mmol and 5 μCi ³ H/10 μl in dilute sulfuricacid/ethanol.

5. Catechol-O-methyl transferase from rat liver--0.45 ml. total volume

Protein concentration of enzyme solution varies from 15 to 40 mg./ml.Solution also contains phosphate buffer, glutathione and dithiothreitol.

6. Stopping Solution--5 ml. total volume

Contains 800 mM boric acid and 80 mM disodium EDTA in 1 N sodiumhydroxide, 4 mM DL-metanephrine HCl, 4 mM DL-normetanephrine HCl, and 4mM methoxytyramine HCl.

7. Sodium metaperiodate 4% (w/v) solution--5 ml. total volume

8. Glycerol 10% (v/v) solution--5 ml. total volume

C. Preparation of Mammalian System

A sample of human blood is drawn from a patient. The blood is collectedinto a tube containing a mixture of EGTA (250 mM) and glutathione (200mM). The solution should have a pH of 5-6. 20 μl. of the solution isadded for each ml. of whole blood collected. Plasma is then obtained bycentrifugation of the blood sample. The plasma is frozen. Care should betaken to free the sample of fibrin and other particulate matter orcloudiness.

D. Reagent Mixture

A reagent mixture is prepared with the following components from B andin the following order and proportions:

    ______________________________________                                        Solution 3         10 μl.                                                  Solution 4         10 μl.                                                  Solution 5         5-20 μl.                                                Distilled Water*                                                              to a total volume of                                                                             40 μl.                                                  ______________________________________                                         *(Deionized, glass distilled)                                            

Vortex the mixture lightly before the enzyme is added and lightly afterthe reagent mixture is complete. Multiples of the 40 μl. reactionmixture are prepared.

E. Standard and Stabilizing Solution

Dilute an aliquot of Solution 2 in a ratio of 1:10,000 with chilleddistilled water and store in an ice bath. This will provide a "workingstandard" of 100 picograms of each catecholamine per 10 μl.

An aliquot of Solution 1 is similarly diluted.

E. Incubation

To a tube containing 50 μl. of the blood serum is added 10 μl. ofSolution 1 and 40 μl. of the reagent mixture of Step D.

A blank, a reference standard, and a plasma sample of knowncatecholamine concentration is run alongside the unknown samples whichare done in duplicate. The reference standard is included to determinethe O-methylating efficiency of the enzyme in water and the absence ofthe mammalian system. The plasma sample of known catecholamines isincluded to determine the quality control of the assay.

The blank is prepared from 50 μl. of distilled water, 10 μl. of Solution1 and 40 μl. of the reagent mixture.

The reference standard is prepared from 50 μl. of distilled water, 10μl. of the working standard of Step E and 40 μl. of the reactionmixture.

The plasma sample of known catecholamine content is prepared from 50 μl.of blood plasma, 10 μl. of working standard of Step E and 40 μl. of thereaction mixture.

The above sample tube containing the unknown and the other solutiontubes are treated the same throughout the assay. However, only referenceto the unknown sample tube shall be made in the following steps.

The sample tube is vortexed lightly and incubated at 37° C. for sixty(60) minutes in an oscillating water bath. After the incubation the tubeis returned to the ice bath and 50 μl. of solution 6 is added.

G. Extraction

Two (2) ml. of toluene:isoamyl alcohol (3:2, volume/volume) are added tothe stopped incubate. The mixture is vigorously vortexed and centrifuged(2 minutes at 2,000 rpm). Subsequent centrifugations are done in thesame manner. Two distinct layers will result. The tube is placed in adry ice-ethanol bath and the aqueous layer frozen. The organic phase isdecanted into a tube containing 0.1 ml. of 0.1 N acetic acid. The tubeand contents are vortexed, centrifuged and frozen in the same manner asbefore. The organic phase is aspirated from the frozen aqueous phase anddiscarded. The frozen aqueous phase is thawed, 1 ml. of toluene:isoamylalcohol (3:2, volume/volume) is added, the tube and contents arevigorously vortexed and centrifuged. The aqueous phase is frozen and theupper organic phase is aspirated and discarded.

H. Separation

All of the acetic acid phase from G is applied to the bottom of asilicagel GF plate 2.5×10 cm, 250 micron layer obtained from Aaltech,Inc. The aqueous spot is dried with the aid of 0.15 ml. of absoluteethanol added to the tube prior to spotting and a warm air flow. Thespotted plate is placed in a paper lined preequilibrated thin layerchromatography developing tank. The plates are developed with a solventsystem of isopropyl alcohol/n-butyl alcohol/water/formic acid,60:20:19:1. The solvent is permitted to move to the top of the plate,approximately one hour. Remove the plate and dry thoroughly. The zonesare visualized under 254 nm ultraviolet light. The upper zone containsNMN and MEOT. The lower zone contains MN. The silica gel of each zone isscraped off the plate and put into different tubes.

I. Oxidation of NMN

To the tube containing the NMN and MEOT, add 1 ml. of 1 N ammoniumhydroxide to elute the amines from the silica gel and vortex vigorously.50 μl. of Solution 7 is added and the tube vortexed. Four minutes afterthe addition of Solution 7, add 50 μl. of Solution 8 and vortex.

J. Extraction and Counting of Vanillin from NMN

100 μl. of glacial acetic acid is added to the tube and vortexed. 10 ml.of toluene/Liquifluor® is added to the tube and the tube vigorouslyshaken for thirty seconds. The tube is centrifuged for two minutes at2000 rpm, the layers allowed to separate and the aqueous layer frozen.The upper organic layer is decanted into a separate scintillation vialcontaining 2 ml. of 0.1 N acetic acid. The vial is capped, shakenvigorously, and counted in a liquid scintillation counter. Theradioactivity in the vial comes from the level of NE in the sample.

K. Extraction and Counting of MEOT

The frozen aqueous layer of J is thawed and 5 ml. of toluene is added.The tube and contents is vortexed, centrifuged for two minutes at 2,000rpm and the aqueous layer frozen. The upper organic phase is aspiratedand discarded. 0.3 ml. of concentrated ammonium hydroxide is added andthe tube vortexed. The pH of the remaining aqueous phase should bebetween 9.8 and 10.1. Ten ml. of toluene:isoamyl alcohol (3:2volume/volume) is added. The tube is vigorously shaken, centrifuged fortwo minutes at 2,000 rpm and the aqueous layer frozen. The upper organicphase is decanted into a scintillation vial containing 10 ml. ofDiotol®, a liquid scintillation medium obtained from Burdick andJackson. The vial is shaken and counted in a liquid scintillationcounter. The radioactivity in the vial comes from the level of DA in thesample.

L. Oxidation of MN, Extraction and Counting of Vanillin from MN

The scrapings of silicagel of the lower zone are treated in the samemanner as in I. The extraction and counting is performed in the samemanner as J. The radioactivity in the vial comes from the level ofepinephrine in the sample.

EXAMPLE 2

In order to perform the non-differentiated assay for total levels of NEand E, the same assay procedure as in Ex. 1 is done except that theseparation step is omitted. The acetic acid phase obtained from thetoluene:isoamyl alcohol (3:2, volume/volume) extract of the stoppedincubate is dried under a stream of air or reduced pressure. 1 ml. of 1N ammonium hydroxide is added to the dried residue and the tube andcontents vortexed. The contents of the tube are then oxidized tovanillin, extracted and counted as in Steps I and J of Example 1. Theradioactivity of the vial comes from the total, undifferentiated levelsof epinephrine and NE in the sample.

EXAMPLE 3

The assays of Examples 1 and 2 are performed on human urine samples orthe supernatants of deproteinized tissue homogenates.

Similar assay results are obtained as in the above examples.

In order to secure a more reproducible and accurate assay level ofdopamine in each of Examples 1, 2 and 3, a DOPA decarboxylase inhibitoris present in the incubation mixture. Compounds which can inhibit theenzyme DOPA decarboxylase are illustratively members of thebenzyloxyamine family and water soluble hydrazines. Illustrativeexamples of the benzyloxyamines are benzyloxyamine,3-hydroxy-4-bromobenzyloxyamine and their acid addition salts, forexample, sulfuric, phosphoric, and hydrochloric. An example of a watersoluble hydrazine is phenylhydrazine.

A further broad aspect of the invention is the assemblage of essentialcomponents of the assay into a kit utilized in laboratories. As statedpreviously, knowledge of catecholamine levels are significant in thediagnosis of a number of conditions. Commercial laboratories, hospitalsand clinics would have a substantial need for this assay. Such a needcould be best filled by having available numerous multiples of thenecessary reagents together with the appropriate instructions concerningthe assay. Such a kit could be readily available by commercial shippingto satisfy the needs of the laboratories, hospitals and clinics. Inaccordance with this invention, there is a container having within it amultiplicity of smaller containers and contents, said contentscomprising the items enzymes catechol-O-methyl transferase, the labeledmethyl donor S-adenosyl-L-methionine(³ H)methyl essentially free ofunlabeled methyl donor, a cation of oxidation number +2 which allows acatechol-O-methyl transferase catalyzed O-methylation of a catecholamineto proceed, a compound which stabilizes acatechol-O-methyl-transferase-catecholamine enzyme-substrate system, andan agent which selectively removes calcium ions from interference with acatechol-O-methyl transferase catalyzed O-methylation of acatecholamine; with the proviso that the S-adenosyl-L-methionine(³H)methyl does not have any other item in its smaller container and withthe overall proviso that each item is of such a quantity that the enzymecatalyzed transfer of methyl from the labeled donor to thecatecholamine, initiated from the bringing together of all the items ata proper pH and temperature and in the presence of a catecholaminesample from a mammalian system selected from the group consisting of thesupernatant of a deproteinized homogenate, blood serum or plasma, and abiological fluid, shall O-methylate (³ H) substantially all of thecatecholamines in the sample.

The large container which holds the smaller containers can be made ofstyrofoam or any other material which is reasonably impact resistant.The styrofoam or other material is solid throughout with holes in thetop wherein the smaller containers, usually circular vials, will snuglyfit. Inside the smaller containers will be contents which will go intothe assay incubate. The labeled methyl donor should not have any otheritem in its container. The enzyme, cation, enzyme-substrate stabilizingcompound, and agent which selectively removes calcium ion can each bewithin its own smaller container or any combination of them within asmall container. Other reagents useful in the assay can also be includedin the kit, for example, the buffer system being used to maintain the pHof the incubate and the incubate stopping solution. The incubatestopping solution should obviously not be present in any smallercontainer whose contents have an incubate component. The largercontainer holding the smaller containers is partially or wholly coveredby a top which is free of the container or attached thereto in somemanner as by a hinge. Within the container or top or attached to thecontainer or top is a means for providing instructions on the assayprocedure. This means can be in the form of a printed sheet, a taperecording, or any other means which conveys the information concerningthe proper procedure for performing the assay.

Because of the relatively unstable nature of some of the reagents, thekit should be kept at a reduced temperature when not in use, preferablybelow -20° C.

It should be noted that the exemplification and preferences which applyto the assay components apply equally as well to the kit components usedin the assay.

Throughout the specification and claims is the expression"O-methylated(³ Hepinephrine and norepinephrine". It is to be understoodthat the norepinephrine is also O-methylated(³ H). A similarinterpretation is to be understood when the (³ H) is not present.

The concentration of enzyme generally used in the incubation is thatamount necessary to obtain maximal velocity with a given amount ofmethyl donor and catecholamine.

Earlier in the specification, the acidic solvent system forchromatographic separation was indicated as being preferred. A newpreference has been established for the basic solvent system,specifically 6 parts t-amylalcohol:2 parts benzene; 3 parts methylaminesolution, volume to volume.

When dealing with a mammalian tissue sample, the tissue must bedisrupted and deproteinized prior to use in the assay. This can be doneby means such as homogenizing, sonication or tissue press underpressure. The solid mass is separated from the liquid by centrifugation.The liquid supernatant is employed in the assay. The phrase "supernatantof the deproteinized tissue homogenate" appearing through thespecification and claims is intended to mean the supernatant of tissuedisrupted by any means, not just a homogenizing technique.

A "tris" buffer is a tris(hydroxymethyl)aminomethane buffer. Such abuffer is used to maintain the pH of the incubate.

When referring throughout the specification to xylenes as a solventwhich extracts the O-methylated(³ H)catecholamines, o-xylene is intendedto be included as well as m- and p-xylene.

Throughout the specification and claims, a component of the assayincubate has been "an agent which preferentially removes calcium ionsfrom interference with the enzymatic reaction". The function of theseagents is to stop the calcium ion from inhibiting the enzymaticreaction. Since enzyme activating cations of oxidation number +2 such asmagnesium, cobalt and manganese are necessary for enzyme activity, it isof course preferred to have an agent which selectively removes thecalcium ion from interference with the enzymatic reaction, such as EGTA,leaving the enzyme activating cation concentrations at a level whereinenzyme activation occurs.

An agent which removes the calcium ion from interference with theenzymatic reaction need not do so preferentially but can still be usedin the assay. For example, many chelating agents will remove significantquantities of magnesium as well as calcium and can be used in the assay.The loss of available enzyme activating cation in the incubate can beovercome by the addition of extra activating cation in sufficientquantity to produce substantial or preferentially optimal enzymeactivity.

Agents of this "nonselective" type which remove calcium frominterference with the enzymatic reaction are illustrativelyethylene(dinitrilo)tetraacetic acid (EDTA), usually as the sodium salt;1,2-diaminocyclohexane N,N,N',N'-tetraacetic acid; andN'(2-hydroxyethyl)ethylene diamine N,N,N'-triacetic acid. Theconcentration of the agent in the assay is from about 2 to about 50 mMof agent, preferably about 5 to about 20 mM.Ethylene(dinitrilo)tetraacetic acid (EDTA) is the preferred compound ofthis category. The quantity of excess activating ion concentrationneeded in the incubate to achieve substantial or maximal enzymeactivation can be readily calculated from knowledge of the bindingcapacity of the particular agent being employed or by simpleexperimentation.

It is to be understood that this nonselective agent which also removesenzyme activating ions as well as calcium ion from interaction with theenzyme can be substituted for the "agent which preferentially removescalcium ions from interference with the enzymatic reaction" in the assayprocedure within the specification and within the claims as well. Thissubstitution can be made in independent claims 1, 23, 63, 64, 93, 101,121 and 126. As an example of this further aspect of the invention isthe composition which comprises the enzyme catechol-O-methyltransferase, a tritium labeled methyl donor S-adenosyl-L-methionine(³H)methyl essentially free of unlabeled methyl donor, a cation ofoxidation number +2 which allows the methyl transfer to proceed,compound which stabilizes the enzyme-substrate system, an agent whichremoves calcium ions from interference with the enzymatic reaction and amammalian system selected from the group consisting of the supernatantof deproteinized tissue homogenate, a blood serum or plasma, and abiological fluid, the transferase, cation, stabilizing compound, methyldonor, and agent which removes calcium ions present in such quantitiesthat substantially all the epinephrine, norepinephrine and dopaminepresent in the mammalian system are O-methylated-(³ H).

We claim:
 1. A method for oxidizing metanephrine or normetanephrine tovanillin which comprises contacting the metanephrine or normetanephrinewith periodate in the contact presence of silica gel.
 2. A method inaccordance with claim 1 wherein the metanephrine or normetanephrine istritium labeled at the methoxy position.
 3. A composition whichcomprises a catecholamine selected from the group consisting ofmetanephrine and normetanephrine, silica gel and periodate.
 4. Acomposition in accordance with claim 3 wherein the catecholamine istritium labeled at the methyl position.